Detection and Molecular Characterization of Adenoviruses in Captive and Free-Roaming African Green Monkeys (Chlorocebus sabaeus): Evidence for Possible Recombination and Cross-Species Transmission

In the present study, 31 samples (12 fecal, 9 nasal and 10 rectal swabs) from 28/92 (30.43%, 10 captive and 18 free-roaming African green monkeys (AGMs, Chlorocebus sabaeus)) apparently healthy AGMs in the Caribbean Island of St. Kitts tested positive for adenoviruses (AdVs) by DNA-dependent DNA polymerase (pol)-, or hexon-based screening PCR assays. Based on analysis of partial deduced amino acid sequences of Pol- and hexon- of nine AGM AdVs, at least two AdV genetic variants (group-I: seven AdVs with a Simian mastadenovirus-F (SAdV-F)/SAdV-18-like Pol and hexon, and group-II: two AdVs with a SAdV-F/SAdV-18-like Pol and a Human mastadenovirus-F (HAdV-F)/HAdV-40-like hexon) were identified, which was corroborated by analysis of the nearly complete putative Pol, complete hexon, and partial penton base sequences of a representative group-I (strain KNA-08975), and -II (KNA-S6) AdV. SAdV-F-like AdVs were reported for the first time in free-roaming non-human primates (NHPs) and after ~six decades from captive NHPs. Molecular characterization of KNA-S6 (and the other group-II AdV) indicated possible recombination and cross-species transmission events involving SAdV-F-like and HAdV-F-like viruses, corroborating the hypothesis that the evolutionary pathways of HAdVs and SAdVs are intermingled, complicated by recombination and inter-species transmission events, especially between related AdV species, such as HAdV-F and SAdV-F. To our knowledge, this is the first report on detection and molecular characterization of AdVs in AGMs.


Introduction
Adenoviruses (AdV), members of the family Adenoviridae, are ubiquitous viruses that have been reported in a wide variety of animal species (mammals, birds, reptiles, amphibians, and fish), and in environmental samples [1][2][3][4][5]. Although many of the AdVs are considered as low-grade pathogens that typically cause asymptomatic or subclinical infections, certain AdVs have been linked to acute clinical disease in humans and other few fecal samples (n = 4) obtained from free-roaming AGMs in Senegal tested negative for AdVs by qPCR and conventional PCR assays [23]. Adenoviruses have been propagated in kidney cell lines derived from AGMs [36], and anti-AdV antibodies have been reported in AGMs [37]. In the present study, based on nasal and fecal/rectal samples from AGMs in St. Kitts, we report for the first-time detection and molecular characterization of AdVs in captive and free-roaming AGMs, providing evidence for possible recombination and cross-species transmission events.

Sampling
The present study was based on samples obtained from 92 apparently healthy AGMs on the island of St. Kitts, and included (i) rectal and nasal swabs from 52 free-roaming AGMs (sampled during September 2022-January 2023), (ii) fecal samples from 5 freeroaming AGMs ( Following trapping, the free-roaming AGMs were sedated with ketamine (dose 7-10 mg/kg, intramuscular injection in hindleg) (Ketamidor ® , Chanelle Pharma, Galway, Ireland), and once the animals reached proper sedation levels, nasal and rectal samples were obtained using sterile swabs (MicroTest™ M4RT, Thermo Fisher Scientific Inc., Waltham, MA, USA) (one each for nasal and rectal sample) from respective orifices, and transferred into separate sterile tubes containing viral transport medium (MicroTest™ M4RT, Thermo Fisher Scientific Inc., Waltham, MA, USA). The fecal samples were obtained from the rectal orifice (using a lubricated sterile glove) of the sedated AGM and transported into a sterile container (Sigma-Aldrich, Milwaukee, WI, USA). Some of the free-roaming AGMs were sampled at the trapping site, whilst others were sampled immediately after transportation to the nearby BSF facility.
On the other hand, samples were obtained from the captive and pet AGMs by scooping a small volume of the material from the top of the fecal pile immediately after the NHP had defecated in the cage. The captive monkeys at BSF were sampled the next day, or a few days after they were trapped. All samples were preserved at −80 • C until further analysis. The procedures for trapping and sampling of AGMs were approved by the Ross University School of Veterinary Medicine (RUSVM), St. Kitts, IACUC 2014 #14-6-032 and IACUC #22-5-11 and followed the RUSVM IACUC approved "Monkey trapping policies and procedures" (SOP # NHP001, April 2022).

Amplification of AdV DNA
Viral DNA was extracted from the AGM samples using the QIAamp DNA Mini Kit (Qiagen Sciences, Germantown, MD, USA). The samples were screened for the presence of AdV DNA using a nested PCR assay that targeted a partial stretch of the AdV pol gene (~250 bp) as described by Roy et al. [38]. Samples that tested negative with the Roy primers were further screened for AdVs with a hexon-based PCR assay as reported by Ba'nyai et al. [8]. Primers used in PCR/semi-nested PCR/nested PCR assays to obtain the nearly complete Pol, complete hexon, and partial penton base coding sequences of the AGM AdVs were designed in the present study and are shown in Supplementary Table S1. The PCR assays were carried out using the Platinum™ Taq DNA Polymerase (Invitrogen™, Thermo Fisher Scientific Corporation, Waltham, MA, USA), or the QIAGEN Hot Star Taq Master Mix Kit (Qiagen Sciences, Germantown, MD, USA) according to the manufacturer's instructions. To rule out contamination issues, sterile water was used as a negative control in all PCR reactions.

Nucleotide Sequencing
The Wizard ® SV Gel and PCR Clean-Up kit (Promega, Madison, WI, USA) was used to purify the PCR products following the protocol outlined by the manufacturer. Nucleotide (nt) sequences were obtained by the Sanger dideoxy chemistry using the ABI Prism Big Dye Terminator Cycle Sequencing Ready Reaction Kit (Applied Biosystems, Foster City, CA, USA) on an ABI 3730XL Genetic Analyzer (Applied Biosystems, Foster City, CA, USA). Nucleotide sequences were obtained in both directions.

Sequence Analysis
Homology search for related AdV sequences was performed using the standard BLASTN and BLASTP program (Basic Local Alignment Search Tool, www.ncbi.nlm.nih. gov/blast, accessed on 20 May 2023). The putative coding regions and corresponding deduced amino acid sequences (aa) were determined using the ORF finder (https: //www.ncbi.nlm.nih.gov/orffinder/, accessed on 15 May 2023), and confirmed by BLASTN and BLASTP analysis, respectively. Pairwise nt and deduced aa sequence identities (%) were calculated using the BLASTN and BLASTP program, respectively, with the 'align two or more sequences' option (accessed 20 May 2023). Multiple alignments were carried out using the CLUSTALW program (https://www.genome.jp/tools-bin/clustalw, accessed on 22 May 2023) with default parameters. Phylogenetic analysis was performed by the maximum likelihood (ML) method using the MEGA11 software [39], with the LG+I+G model of substitution and 1000 bootstrap replicates, as described previously [1,3]. Recombination analysis was carried out using the RDP4 program [40], with parameters described previously [41]. The 'G + C' content (%) of AdV sequences were determined using the GC Content Calculator (https://www.biologicscorp.com/tools/GCContent/, accessed 20 May 2023).

GenBank Accession Numbers
The GenBank accession numbers for the AGM AdV sequences determined in this study are OR066890-OR066927.

Detection of AdVs in AGMs
The sampling/trapping sites for the captive (at the BSF quarantine facility) and freeroaming AGMs are shown in Figure 1. Since the pan-pol nested PCR assay described by Wellehan et al. [42] has been used to detect a wide variety of AdVs, especially novel AdVs in wildlife [43], a subset of AGM samples (n = 20) from the present study were initially screened using the Wellehan primers. Seven samples yielded the expected~300 bp amplicon, which were shown to be amplification of non-AdV genomes by sequencing of the PCR products followed by BLASTN analysis. These observations, and similar findings from other studies (Gainor et al. [44], and 33 of 37 fecal/rectal samples from bats on St. Kitts tested false positive for AdVs) raise concerns on the specificity of the Wellehan primers in AdV screening assays.
In the present study, all the AGM samples from St. Kitts were screened by a polbased nested PCR assay that has been used to detect AdVs in NHPs [23,25,38]. A total of 28 samples (9 fecal, 9 nasal and 10 rectal samples) tested positive for AdVs using the pol PCR assay. Furthermore, screening of the pol PCR negative samples with a hexon-based PCR assay [8] revealed the presence of AdVs in 3 additional fecal samples. All screening results were confirmed by sequencing (and BLASTN analysis) of the PCR amplicons. The details of the AGMs that tested positive for AdVs are shown in Table 1. Overall, 31 samples (12 fecal, 9 nasal and 10 rectal) from 28 AGMs (30.43% (n = 92), 3 animals tested positive for both nasal and rectal swabs) tested positive for the presence of AdV DNA. The rates of detection of AdVs in nasal, fecal, and rectal samples were 17.3% (9/52 AGMs), 30% (12/40), and 19.2% (10/52), respectively. The AdV detection rates in captive, freeroaming (trapped), and caged pet AGMs were 33.33% (10/30 AGMs), 31.57% (18/57), and 0% (0/5), respectively. Although the sample size from free-roaming AGMs was almost twice of that from captive AGMs, the AdV detection rates were comparable between both groups. Since the samples were obtained from captive AGMs in quarantine cages the next day, or a few days after trapping, it is more likely that they acquired the infection before introduction into the BSF facility. All the AdV positive AGMs were apparently healthy at the time of sampling. In the present study, all the AGM samples from St. Ki s were screened by a pol-base nested PCR assay that has been used to detect AdVs in NHPs [23,25,38]. A total of 28 sam ples (9 fecal, 9 nasal and 10 rectal samples) tested positive for AdVs using the pol PC assay. Furthermore, screening of the pol PCR negative samples with a hexon-based PC assay [8] revealed the presence of AdVs in 3 additional fecal samples. All screening resul were confirmed by sequencing (and BLASTN analysis) of the PCR amplicons. The detai of the AGMs that tested positive for AdVs are shown in Table 1. Overall, 31 samples (1 fecal, 9 nasal and 10 rectal) from 28 AGMs (30.43% (n = 92), 3 animals tested positive fo both nasal and rectal swabs) tested positive for the presence of AdV DNA. The rates o detection of AdVs in nasal, fecal, and rectal samples were 17.3% (9/52 AGMs), 30% (12/40 and 19.2% (10/52), respectively. The AdV detection rates in captive, free-roamin (trapped), and caged pet AGMs were 33.33% (10/30 AGMs), 31.57% (18/57), and 0% (0/5 respectively. Although the sample size from free-roaming AGMs was almost twice of tha from captive AGMs, the AdV detection rates were comparable between both groups. Sinc the samples were obtained from captive AGMs in quarantine cages the next day, or a few days after trapping, it is more likely that they acquired the infection before introductio into the BSF facility. All the AdV positive AGMs were apparently healthy at the time o sampling. Green hill 3 1 The AGM was sampled at the trapping site. 2 The fecal samples were obtained from captive AGMs (housed in individual cages) at the quarantine facility of Behavioral Science Foundation (BSF), Estridge estate, St. Kitts. 3 The AGM was sampled immediately after arrival to the BSF quarantine facility from the trapping site 3 .

Analysis of the Partial Pol and Hexon Sequences of AGM AdV Strains
By BLASTN analysis, the AdV partial pol sequences (n = 25,~200 bp after trimming end sequences, from the pol-based screening PCR assay [38]) from AGMs shared~98-100% nt identities between themselves, and 95.5-98.9% identities with cognate pol sequences of SAdV-F/SAdV-17 (Mastadenovirus species/AdV serotype, or genotype) strain B-105 (Gen-Bank accession number KP329566) and SADV-F/SAdV-18 strain C676 (FJ025931) from grivet monkeys (Chlorocebus aethiops) [1,[45][46][47][48][49], followed by identities of 84.54-88.83% with those of other AdVs. Although the remaining partial pol sequences (n = 3) and all the 3 AdV partial hexon sequences (~250 bp after trimming end sequences, from the hexonbased screening PCR assay [8]) shared maximum homology with SADV-F/SAdV-18, they lacked quality (phred value < 40) and were not considered for sequence identities. Between themselves, the pol of SAdV-F/SAdV-17 and -18 are highly conserved (97.4% nt sequence identities), whilst their hexon are genetically divergent (83.20% identities). Since the AGM AdV partial pol sequences were closely related to both SAdV-F/SAdV-17 and -18, 24 of the 31 AdV positive samples were subjected to a semi-nested PCR assay (designed in this study, Supplementary Table S1) targeting a partial stretch of the hexon (corresponding to nt 18458-nt 19307 of SAdV-F/SAdV-18) that is genetically divergent between SAdV-F/SAdV-17 and -18. Seven samples were not included, as they exhibited faint amplification with the pol-based screening PCR assay and were not available in sufficient volumes. Out of the 24 samples, 9 tested positive with the hexon-based semi-nested PCR assay, which included only one of the 3 samples that were positive for the hexon-based screening PCR assay. Surprisingly, the 13 pol PCR positive AdV samples, negative for the hexon-based semi-nested PCR assay, also tested negative with the hexon-based screening PCR assay. A flow chart summarizing the work pipeline and the outcomes from different PCR assays is shown in Supplementary Figure S1.
Based on nt sequence identities, the partial hexon sequences (~750 bp after trimming end sequences, from hexon-based semi-nested PCR) of the nine AGM AdV strains were classified into two groups, consisting of seven (group-I) and two AdV (group-II) strains, respectively. Members within each group shared 100% nt sequence identities between themselves, whilst nt and deduced aa identities of~67% and~70%, respectively, were observed between the groups. Interestingly, a single AGM (KNA-2-22) tested positive for both group-I (nasal swab) and -II (rectal swab) viruses. The partial hexon aa sequences (~250 aa) of group-I viruses shared maximum identities of~88% with cognate sequences of SAdV-F/SAdV-18 and <83% identities with other AdVs, whilst group-II viruses shared maximum identities of~91% with AdV strain GgorAdV-1 (GenBank accession number AEM45085) from a gorilla (Gorilla gorilla) [19], followed by~86% identities with AdV strain PtroAdV-3 (JN163973) from a chimpanzee (Pan troglodytes) [19] and some HAdV-F/HAdV-40 strains. These findings were corroborated by phylogenetic analysis of the partial hexon aa sequences ( Figure 2). Mastadenovirus species HAdV-F (consisting of HAdV-40 and -41) and SAdV-F (SAdV-17 and -18) are considered as sister taxons [17], whilst SAdV strains GgorAdV-1 and PtroAdV-3 were shown to be closely related to HAdV-F [19], and strain PtroAdV-3 was proposed to be transmitted from humans to a chimpanzee [19]. Of note here, a third SAdV strain, ROC2012 (MN136539, from a moustached guenon, Cercopithecus cephus) was also shown to cluster with HAdV-F AdVs [50]. However, only a 275 nt hexon sequence was available for ROC2012 in the GenBank database, which was not sufficient for analysis. Based on nt sequence identities, the partial hexon sequences (~750 bp after trimming end sequences, from hexon-based semi-nested PCR) of the nine AGM AdV strains were classified into two groups, consisting of seven (group-I) and two AdV (group-II) strains, respectively. Members within each group shared 100% nt sequence identities between themselves, whilst nt and deduced aa identities of ~67% and ~70%, respectively, were observed between the groups. Interestingly, a single AGM (KNA-2-22) tested positive for both group-I (nasal swab) and -II (rectal swab) viruses. The partial hexon aa sequences (~250 aa) of group-I viruses shared maximum identities of ~ 88% with cognate sequences of SAdV-F/SAdV-18 and <83% identities with other AdVs, whilst group-II viruses shared maximum identities of ~91% with AdV strain GgorAdV-1 (GenBank accession number AEM45085) from a gorilla (Gorilla gorilla) [19], followed by ~86% identities with AdV strain PtroAdV-3 (JN163973) from a chimpanzee (Pan troglodytes) [19] and some HAdV-F/HAdV-40 strains. These findings were corroborated by phylogenetic analysis of the partial hexon aa sequences ( Figure 2). Mastadenovirus species HAdV-F (consisting of HAdV-40 and -41) and SAdV-F (SAdV-17 and -18) are considered as sister taxons [17], whilst SAdV strains GgorAdV-1 and PtroAdV-3 were shown to be closely related to HAdV-F [19], and strain PtroAdV-3 was proposed to be transmi ed from humans to a chimpanzee [19]. Of note here, a third SAdV strain, ROC2012 (MN136539, from a moustached guenon, Cercopithecus cephus) was also shown to cluster with HAdV-F AdVs [50]. However, only a 275 nt hexon sequence was available for ROC2012 in the GenBank database, which was not sufficient for analysis. Taken together, the partial Pol sequences of group-I and -II AGM AdV strains were closely related to both SAdV-17 and -18, whilst the partial hexon sequences of group-I and -II viruses were more related to those of SAdV-F/SAdV-18 and HAdV-F/HAdV-40 (and Taken together, the partial Pol sequences of group-I and -II AGM AdV strains were closely related to both SAdV-17 and -18, whilst the partial hexon sequences of group-I and -II viruses were more related to those of SAdV-F/SAdV-18 and HAdV-F/HAdV-40 (and SAdVs from a gorilla and a chimpanzee that were closely related to HAdV-F), respectively, than those of other AdVs (Figure 2). Considering these observations, two AGM AdV strains, KNA-S6 and KNA-08975, representing group-I and -II viruses, respectively, and available in sufficient volumes, were selected for further molecular characterization.  Table S1) and 2 reverse primers (designed from the obtained penton base sequences of KNA-S6 and KNA-08975) in separate PCR assays failed. Since SAdV strains GgorAdV-1 and PtroAdV-3 (related to HAdV-F) have been characterized for partial genomes [19], they were excluded from further analysis. In the present study, the analyses of the putative Pol, hexon and penton base of KNA-S6 and KNA-08975 were based on deduced aa sequences, as reported in previous studies [1,3,16], and the outcomes were similar to those observed with nt sequences. To rule out biases, phylogenetic analyses were performed with both the ML method and the Neighbor-Joining method, revealing similar clustering patterns.
The base composition ('G + C' %) of viral genomes has been considered as an a tional parameter for classification of AdV species, and the 'G + C' content of partial A Based on analyses of deduced aa sequences of the three major AdV proteins (Pol, hexon and penton base), AGM AdV KNA-08975 was shown to be more closely related to SAdV-F/SAdV-18 (SAdV-F/SAdV-18-like) than those of other AdVs. On the other hand, the origin of KNA-S6 appear to be complex, with a SAdV-F/SAdV-18-like Pol, and a hexon and a penton base that was more closely related to HAdV-F/HAdV-40(HAdV-F/HAdV-40-like) and SAdV-F/SAdV-17, respectively. The AdV hexon has been proposed to be an active site for recombination events [3,[14][15][16][17][18]. Although recombination analysis should be performed on complete genomes of AdVs [17], we attempted to evaluate the complete hexon coding sequence of KNA-S6 (and KNA-08975) for possible recombination events. However, inconclusive results were obtained with the RDP4 program.

Discussion
To our knowledge, this is the first report on detection and molecular characterization of AdVs from AGMs. Overall, high AdV detection rates (30.43%, 28/92 AGMs) were observed in the AGMs from St. Kitts. Based on analysis of the partial Pol-and hexon-aa sequences of a subset of AGM AdVs, and the nearly complete Pol, complete hexon and partial penton base sequences of two representative AdV strains (KNA-08975, group-I and KNA-S6, group-II), it appears that at least two AdV genetic variants (group-I: 7 AdVs with a SAdV-F/SAdV-18-like Pol and hexon, and group-II: 2 AdVs with a Pol and a hexon more closely related to that of SAdV-F/SAdV-18 and HAdV-F/HAdV-40, respectively, than other AdVs) might be circulating and even endemic (as both AdV variants were detected in samples from 2014-2015 and 2022) to the AGM population on St. Kitts. Several of the AdVs with a SAdV-F/SAdV-17/18-like partial Pol could not be amplified for the hexon gene, indicating that the levels of genetic diversity among AGM AdVs in St. Kitts might be higher than those reported in the present study.
The mastadenovirus species SAdV-F is understudied, represented by only two isolates (SAdV-17 and -18) that were detected in captive grivet monkeys during the 1950s [1,45,[47][48][49], and since then, there has been no published reports on the identification of these viruses from NHPs (based on a PubMed search using keywords 'simian/monkey/non-human primate/non-human primate/non-human primate adenovirus 17/18', 'adenovirus 17/18', and/or 'simian adenovirus F'). We reported SAdV-F-like AdVs for the first time in freeroaming NHPs and after~six decades from captive NHPs. The detection of SAdV-Flike AdVs and SAdV-F/SAdV-17/18 in related host species (AGMs and grivet monkeys, respectively, genus Chlorocebus) mirrored the hypothesis that AdVs have a narrow host range [1][2][3][4]14,15,17]. SAdV-F/SADV-17 and -18 were detected in the intestinal contents of grivet monkeys [47], whilst the SAdV-F-like AdVs from AGMs were identified in both fecal/rectal and nasal samples. There is a lack of information on the pathogenesis of SAdV-F/SAdV-17 and -18 [17,46], whilst the SAdV-F-like AdVs from this study were detected in apparently healthy AGMs, warranting studies on the tropism and disease-causing potential of SAdV-F in NHPs. The complete genomes of SAdV-17 and -18 were obtained from AdV isolates that have been propagated/maintained in cell cultures for years before they were sequenced [45,46]. On the other hand, the AdV genomes reported in this study were determined from wild-type AdVs detected in AGMs. Interestingly, the putative Pol and hexon sequences of SAdV-F/SAdV-18-like AdV KNA-08975 were 9 aa shorter and 6 aa longer, respectively, than those of SAdV-F/SAdV-18 (cell culture adapted strain C676 [45]).
Interestingly, analyses of the putative Pol, hexon and penton base sequences of AGM AdV group-II strain KNA-S6 (and partial Pol and hexon sequences of KNA-2-2022-rectal) indicated possible recombination and cross-species transmission events involving HAdV-F-and SAdV-F-like viruses. The group-I (KNA-08975) and -II (KNA-S6) representative samples were also screened with HAdV-F-and SAdV-F-specific hexon primers, respectively, ruling out mixed infections with viruses of the other group (Supplementary Table S1). KNA-S6 was detected in a free-roaming AGM trapped and sampled at the Southeastern peninsula of St. Kitts (Table 1). Although this area is not inhabited by humans, there are a few restaurants by the beach, and a harbor was being constructed at the time of trapping, which might have resulted in possible NHP-human contact. Furthermore, humans have been known to let loose their pet animals in this part of the island, and therefore, KNA-S6 could have been an abandoned pet monkey. On the other hand, KNA-2-2022-rectal was from a captive AGM at the BSF quarantine facility (Table 1). Since the AGM was sampled the next day after trapping, and was in quarantine during sampling, it is likely that the NHP acquired the virus outside the facility. The small Island of St. Kitts has a large population of AGMs (~40,000 [27][28][29]) that are in constant movement, often straying into human habitats, which might have facilitated cross-species transmission events of AdVs.
Although the present study provided first-time and important insights into AdVs circulating in captive and free-roaming AGMs, with evidence for possible recombination and interspecies transmission events involving HAdVs and SAdVs, there were limitations: (i) the study was based on a small sample size (92 AGMs); (ii) sampling lacked consistency (40 fecal samples from AGMs during 2014-2015, whilst nasal and rectal samples from 52 AGMs in 2022); (iii) although the AGM samples were screened using a pol-based nested PCR assay and a hexon-based PCR assay (both assays have been shown to be broadspectrum for NHP AdVs [8,23,25,38]), revealing high detection rates (30.43%, n = 92 AGMs), these screening assays may not be as sensitive as qPCR assays; (iv) since samples KNA-S6 and KNA-08975 were not available in sufficient volumes, we could not determine the whole genomic sequences, or those of other important AdV genes (VA-RNA gene and the fiber gene) for both AdV strains. Of note, HAdV-F, SAdV-F/SAdV-17 and SAdV-F/SAdV-18 have been shown to possess 2, 3, and 1 fiber genes, respectively [46], and (v) we did not have access to samples from humans residing near the AGM trapping sites. Considering the detection of AGM AdVs with a SAdV-F-like Pol and HAdV-F-like hexon, it would have been interesting to investigate the local human population for AdVs.

Data Availability Statement:
The data presented in this study are available in this article and Supplementary Table S1 and Figures S1-S9.